Pigment Epithelium-Derived Factor Protects Retinal Neural Cells and Prevents Pathological Angiogenesis in an Ex Vivo Ischemia Model.

Ocular ischemia/hypoxia is a severe problem in ophthalmology that can cause vision impairment and blindness. However, little is known about the changes occurring in the existing fully formed choroidal blood vessels. We developed a new whole organ culture model for ischemia/hypoxia in rat eyes and investigate the effects of pigment epithelium derived factor (PEDF) protein on the eye tissues. The concentration of oxygen within the vitreous was measured in the enucleated rat eyes and living rats. Then, ischemia was mimicked by incubating the freshly enucleated eyes in medium at 4°C for 14 h. Eyes were fixed immediately after enucleation or were intravitreally injected with PEDF protein or with vehicle before incubation. After incubation, light and electron microscopy (EM) as well as Tunel staining was performed. In the living rats, the intravitreal oxygen concentration was on average at 16.4% of the oxygen concentration in the air and did not change throughout the experiment whereas it was ca. 28% at the beginning of the experiment and gradually decreased over time in the enucleated eyes. EM analysis revealed that the shape of the choriocapillaris changed dramatically after 14 h incubation in the enucleated eyes. The endothelial cells made filopodia-like projections into the vessel lumen. They appeared identical to the labyrinth capillaries found in surgically extracted choroidal neovascular membranes from patients with wet age-related macular degeneration (AMD). These filopodia-like projections nearly closed the vessel lumen and showed open gaps between neighboring endothelial cells. PEDF significantly inhibited labyrinth capillary formation and kept the capillary lumen open. The number of TUNEL-positive ganglion cells and inner nuclear layer cells was significantly reduced in the PEDF-treated eyes compared to the vehicle-treated eyes. The structural changes in the chroidal vessels observed under ischemia/hypoxia conditions can mimic early changes in the process of pathological angiogenesis as observed in wet AMD patients. This new model can be used to investigate short-term drug effects on the choriocapillaris after ischemia/hypoxia and it highlighted the potential of PEDF as a promising candidate for treating wet AMD.

A new rat model of treatment-naive quiescent choroidal neovascularization induced by human VEGF165 overexpression.

Vascular endothelial growth factor (VEGF) is a crucial stimulator for choroidal neovascularization (CNV). Our aim was to develop a reproducible and valid treatment-naive quiescent CNV (i.e. without signs of exudation and with normal visual acuity) rat model by subretinal injection of an adeno-associated virus (AAV)-VEGFA165 vector. The CNV development was longitudinally followed up in vivo by scanning laser ophthalmoscopy/optical coherence tomography, fluorescein and Indocyanine Green angiographies and ex vivo by electron microscopy (EM) and immunohistochemistry. In total, 57 eyes were analysed. In vivo, a quiescent CNV was observed in 93% of the eyes 6 weeks post-transduction. In EM, CNV vessels with few fenestrations, multi-layered basement membranes and bifurcation of endothelial cells were observed sharing the human CNV features. Human VEGF overexpression, multi-layered retinal pigment epithelium (RPE) (RPE65) and macrophages/activated microglia (Iba1) were also detected. In addition, 19 CNV eyes were treated for up to 3 weeks with bevacizumab. The retinal and CNV lesion thickness decreased significantly in bevacizumab-treated CNV eyes compared with untreated CNV eyes 1 week after the treatment. In conclusion, our experimental CNV resembles those seen in patients suffering from treatment-naive quiescent CNV in wet age-related macular degeneration (AMD), and responds to short-term treatment with bevacizumab. Our new model can, therefore, be used to test the long-term effect of new drugs targeting CNV under precisely-defined conditions.

The radioprotector ortho-phospho-L-tyrosine (pTyr) attenuates the side effects of fractionated irradiation in retinoblastoma mouse models but also decreases the local tumour control.

BACKGROUND: Radiotherapy (RT) is used to treat retinoblastoma (Rb), the most frequent ocular tumour in children. Besides eradicating the tumour, RT can cause severe side effects including secondary malignancies. This study aimed to define whether the radioprotector ortho-phospho-L-tyrosine (pTyr) prevents RT-induced side effects and affects local tumour control in a xenograft and a genetic orthotopic Rb mouse model. METHODS: B6;129-Rb1tm3Tyj/J (Rb+/-) and Y79-Rb cell-xenografted nude mice were fractionated external beam irradiated (15 fractions of 5Gy 6MV photons during 3weeks) with or without pTyr pre-treatment (100mg/kg BW, 16h prior to each irradiation). One, three, six and nine months after RT, tumour control and RT toxicity were evaluated using in vivo imaging and histology. We also analysed pTyr dependant post irradiation cell survival and p53 activity in vitro. RESULTS: In vitro pTyr pre-treatment showed no radioprotection on Y79 cells, but led to p53 stabilisation in unirradiated Y79 cells and to a facilitation of radiation-induced p21 up-regulation, confirming a modulation of p53 activity by pTyr. In both mouse models, secondary tumours were undetectable. In Rb+/- mice, pTyr significantly lowered RT-induced greying of the fur, retinal thickness reduction and photoreceptor loss. However, in the xenografted Rb model, pTyr considerably decreased RT-mediated tumour control, which was observed in 16 out of 22 control eyes but in none of the 24 pTyr treated eyes. CONCLUSIONS: In Rb+/- mice pTyr significantly prevents RT-induced greying of the fur as well as retinal degeneration. However, since non-irradiated control mice were not used in our study, a formal possibility exists that the effect shown in the retina of Rb+/- mice may be due to ageing of the animals and/or actions of pTyr alone. Unfortunately, as tested in a xenograft model, pTyr treatment reduced the control of Rb tumours.

Effects of intravitreally injected Fc fragment on rat eyes.

PURPOSE: Anti-vascular endothelial growth factor (VEGF) drugs are used to treat neovascular eye diseases. Some of these drugs contain Fc fragments (Fc), but it is unknown how their mode of action is influenced by Fc. Therefore, this study investigated the effects of Fc on rat eyes after intravitreal injection. METHODS: Eighteen Long-Evans rats were intravitreally injected with sterile, biotin-labeled rat Fc (9.1 µg in 5 µl PBS). For control, 5 µl PBS was injected in another nine rats. Animals were sacrificed between 1 and 3 days (group 1), 7 days (group 2), and 14 days (group 3) after injection. The right eyes were examined by electron microscopy (EM). The left eyes were stained by immunohistochemistry to investigate the distribution of Fc and the presence of macrophages. RESULTS: After 1 day, Fc had penetrated into the anterior chamber and the retina up to the inner nuclear layer, and was located especially in retinal vessels. High numbers of infiltrating cells were present within the vitreous, around the ciliary body, anterior chamber and inside the retina 1-3 days after Fc injection (p < 0.02 group 1 vs. control). Immunohistochemistry and EM showed that they were macrophages or granulocytes in close association with Fc. Ultrastructurally, there were effects on the blood vessels such as thrombocyte activation and fibrin formation. CONCLUSIONS: Biotin labeling is ideal for investigating the distribution of intravitreally injected proteins in ocular tissue. Fc fragments at a dose corresponding to their concentration in standard AMD treatments induced inflammation, and particularly the attraction of immune-competent cells. This may be associated with the risk of inflammation or endophthalmitis after anti-VEGF treatment, and needs further investigation.

Establishment of a novel retinoblastoma (Rb) nude mouse model by intravitreal injection of human Rb Y79 cells - comparison of in vivo analysis versus histological follow up.

Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining. Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patient's tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies.

The effects of VEGF-A-inhibitors aflibercept and ranibizumab on the ciliary body and iris of monkeys.

PURPOSE: To investigate the effects of intravitreal ranibizumab (Lucentis®) and aflibercept (Eylea®) on the ciliary body and the iris of 12 cynomolgus monkeys with regard to the fenestrations of their blood vessels. MATERIALS AND METHODS: Structural changes in the ciliary body and in the iris were investigated with light, fluorescent, and transmission electron microscopy (TEM). The latter was used to specifically quantify fenestrations of the endothelium of blood vessels after treatment with aflibercept and ranibizumab. Each of the two ciliary bodies treated with aflibercept and the two treated with ranibizumab and their controls were examined after 1 and 7 days respectively. Ophthalmological investigations including funduscopy and intraocular pressure measurements were also applied. RESULTS: Ophthalmological investigations did not reveal any changes within the groups. Both drugs reduced the VEGF concentration in the ciliary body pigmented epithelium. The structure of the ciliary body was not influenced, while the posterior pigmented epithelium of the iris showed vacuoles after aflibercept treatment. Ranibizumab was mainly concentrated on the surface layer of the ciliary epithelium, in the blood vessel walls and the lumen of some of the blood vessels, and in the cells of the epithelium of the ciliary body. Aflibercept was more concentrated in the stroma and not in the cells of the epithelium, but as with ranibizumab, also in the blood vessel walls and some of their lumina, and again on the surface layer of the epithelium. Both aflibercept-and ranibizumab-treated eyes showed a decreased number of fenestrations of the capillaries in the ciliary body compared to the untreated controls. On day 1 and day 7, aflibercept had fewer fenestrations than the ranibizumab samples of the same day. CONCLUSIONS: Both aflibercept and ranibizumab were found to reach the blood vessel walls of the ciliary body, and effectively reduced their fenestrations. Aflibercept might eliminate VEGF to a greater extent, possibly due to a higher elimination of fenestrations in a shorter time. Moreover, the vacuoles found in the iris need further research, in order to evaluate whether they carry a possible pathological potential.